FIELD: medicine.
SUBSTANCE: invention relates to medical microbiology. Essence of the proposed invention is the usage of 14 genes, optimally determining the genotype of nontoxigenic strains of V. cholerae of O1 serogroup, for PCR, namely: rstA, tcpA, int, nanH, vce, rtxC, acd-rtxA, vcsN, vspD, vasK, pbd-vgrG3, acd-vgrG1, mshA, stn/sto. Reaction is carried out separately for each gene in 10 mcl of the mixture with primers corresponding to a specific gene and amplification is carried out on a programmable multi-channel thermostat under certain conditions. After completion of the reaction, stain the mixture with ethidium bromide and separate electrophoretically in a 2 % agarose gel prepared on tris-acetate or tris-borate buffer, then read in the transilluminator the length of the amplificate of each fragment, which have the following dimensions: rstA – 1,009 bp, tcpA – 471 bp, int – 458 bp, nanH – 585 bp, vce – 1,009 bp, rtxC – 417 bp, acd-rtxA – 660 bp, vcsN – 508 bp, vspD – 422 bp, vasK – 614 bp, pbd-vgrG3 – 422 bp, acd-vgrG1 – 735 bp, mshA – 432 bp, stn/sto – 172 bp, analyze the results by comparing the isolated determinants of the nucleotide sequences of the genes of the test strain with the length of the amplificates of the control strains: V. cholerae O1 No. M – 878 and No. 14863, as well as the PCR amplificate of this gene obtained on the DNA template of the strain V. cholerae nonO1/non O139 (NRT 36), then compare the obtained result with the tabulated values of the general characteristic of the genotypes of nontoxigenic strains of serogroup O1, on which the identity of the genotype is determined. Reaction mixture for PCR has the following composition: 10-x buffers for amplification (pH 8.8) – 1 mcl; 2.5 mM solution of deoxynucleotide triphosphate mixture (dNTR) – 1 mcl; 2.5 mM solution of direct and reverse primers of a specific gene – 1 mcl; DNA-matrix of the studied strain – 1 mcl; TAQ polymerase (5 U/mcl) 0.1 mcl; deionized water – 6.9 mcl. In addition, carry out the amplification under the following regimes: to determine genes rstA, ACD-rtxA, tcpA, int, nanH vce, mshA, vasK, acd-vgrG1, pbd-vgrG3: denaturation – 94 °C, 3 min (1 cycle); annealing – 58 °C, 30 sec; synthesis – 72 °C, 30 sec (30 cycles); post-synthesis – 72 °C, 3 min (1 cycle); to determine the gene rtxC, vcsN2, vspD: denaturation – 94 °C, 3 min (1 cycle); annealing – 55 °C, 30 sec; synthesis – 72 °C, 30 sec (30 cycles); post-synthesis – 72 °C, 3 min (1 cycle); to determine the stn|sto gene: denaturation – 94 °C, 3 min (1 cycle); annealing – 50 °C, 30 sec; synthesis – 72 °C, 30 sec (30 cycles); post-synthesis – 72 °C, 3 min (1 cycle).
EFFECT: invention can be used to study of nontoxigenic strains of cholera vibrios of different origin at the molecular-biological level with the aim of establishing their role in the etiology of sporadic cases and outbreaks of diarrheal diseases.
3 cl, 6 tbl, 3 ex
